Abstract - The bovine viral diarrhea virus (BVDV) strains exist as two biotypes, cytopathic (cp) and noncytopathic (ncp), according to
their effects on tissue culture cells. It has been previously reported that cell death associated to cp BVDV in vitro is mediated
by apoptosis. Here, experiments were conducted to determine the involvement of the NS3 protein in the induction of apoptosis.
The NS3- and NS3
50 (deleted from the NH2-terminal 50 amino acids)-cDNA encoding sequences of BVDV NADL cp reference strain were cloned into
adenoviral vectors (AdV) from which the BVDV gene of interest could be expressed from a tetracycline-responsive promoter.
A549tTA cells infected in vitro with NS3 or NS3
50-expressing AdV showed cytopathic changes characterized by cell rounding and detachment, and nucleus chromatin condensation.
DNA fragmentation assays, cytochrome c release, and activation of cellular caspases performed on these infected cells clearly
correlated with the observed cytopathic changes with apoptosis. The BVDV NS3
50-induced apoptotic process was inhibited by caspase-8- and -9-specific peptide inhibitors (Z-IETD-FMK and Z-LEHD-FMK). Furthermore,
apoptosis was inhibited in cells expressing the R1 subunit of herpes simplex virus type 2 ribonucleotide reductase (HSV2-R1)
or hsp70, two proteins which are known to inhibit apoptosis associated with caspase-8 activation and cytochrome c release-dependent
caspase-9 activation, respectively. Given that HSV2-R1, a specific inhibitor of the caspase-8 activation pathway, efficiently
suppressed apoptosis and also prevented caspase-9 activation, the overall results indicate that the BVDV NS3/NS3
50 induces apoptosis initiated by caspase-8 activation and subsequent cytochrome c release-dependent caspase-9 activation.